Replacement of the Endogenous Starch DebranchingEnzymes ISA1 and ISA2 of Arabidopsis with the RiceOrthologs Reveals a Degree of Functional Conservationduring Starch SynthesisSebastian Streb*, Samuel C. Zeeman
Institute for Agricultural Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland
Abstract
This study tested the interchangeability of enzymes in starch metabolism between dicotyledonous and monocotyledonousplant species. Amylopectin - a branched glucose polymer - is the major component of starch and is responsible for its semi-crystalline property. Plants synthesize starch with distinct amylopectin structures, varying between species and tissues. Thestructure determines starch properties, an important characteristic for cooking and nutrition, and for the industrial uses ofstarch. Amylopectin synthesis involves at least three enzyme classes: starch synthases, branching enzymes and debranchingenzymes. For all three classes, several enzyme isoforms have been identified. However, it is not clear which enzyme(s) areresponsible for the large diversity of amylopectin structures. Here, we tested whether the specificities of the debranchingenzymes (ISA1 and ISA2) are major determinants of species-dependent differences in amylopectin structure by replacingthe dicotyledonous Arabidopsis isoamylases (AtISA1 and AtISA2) with the monocotyledonous rice (Oryza sativa) isoforms.We demonstrate that the ISA1 and ISA2 are sufficiently well conserved between these species to form heteromultimericchimeric Arabidopsis/rice isoamylase enzymes. Furthermore, we were able to reconstitute the endosperm-specific riceOsISA1 homomultimeric complex in Arabidopsis isa1isa2 mutants. This homomultimer was able to facilitate normal rates ofstarch synthesis. The resulting amylopectin structure had small but significant differences in comparison to wild-typeArabidopsis amylopectin. This suggests that ISA1 and ISA2 have a conserved function between plant species with a majorrole in facilitating the crystallization of pre-amylopectin synthesized by starch synthases and branching enzymes, but alsoinfluencing the final structure of amylopectin.
Citation: Streb S, Zeeman SC (2014) Replacement of the Endogenous Starch Debranching Enzymes ISA1 and ISA2 of Arabidopsis with the Rice Orthologs Revealsa Degree of Functional Conservation during Starch Synthesis. PLoS ONE 9(3): e92174. doi:10.1371/journal.pone.0092174
Editor: Joerg Fettke, University of Potsdam, Germany
Received September 21, 2013; Accepted February 20, 2014; Published March 18, 2014
Copyright: � 2014 Streb, Zeeman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was funded by the Swiss National Science Foundation (Grant Nos. 3100AO-116434/1 and 31003A-131074) and ETH Zurich. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Starch is the major storage carbohydrate in plants and an
important renewable resource for both the food and non-food
industry sectors. Starch is comprised of two glucose polymers
(amylopectin and amylose) and accumulates in plant tissues as
semi-crystalline granules. Amylopectin accounts for the majority of
the granule mass (around 60% to 90%, depending on the
botanical source). It is a branched molecule in which a-1,4-linked
glucan chains are connected via a-1,6-bonds [1,2] resulting in a
tree-like structure. On average, there is one branch point every
20–25 glucose residues. However, the arrangement of branch
points is thought to be non-random, such that linear chain
segments can align together to form double helices that pack into
stable, semi-crystalline lamellae. The branch points are concen-
trated in the amorphous regions between these crystalline lamellae
[1].
The higher-order structures adopted by amylopectin are
thought to occur in all wild-type starches and underlie the
water-insoluble granular characteristics of starch. Nevertheless,
there is considerable variation in starch granule morphology,
structure and composition between plant sources. These factors
are important due to the impact they have on starch properties,
which are relevant for downstream functional applications. A
detailed understanding of how different biosynthetic enzymes
influence amylopectin structure has been hindered by the fact that
it is currently not possible to determine the exact structure of
amylopectin. Also, we can only partially relate the physical
properties of starch to its structure, and hence it is difficult to
predictably control these characteristics through manipulation of
enzyme abundance and/or specificities.
Starch synthesis is mediated by three enzyme classes. Starch
synthases extend a-1,4-linked chains by transferring new glucose
units from ADP-glucose to the non-reducing end. Branch points
are introduced by branching enzymes, which cleave an existing a-
1,4-bond of a linear chain and transfer the cut end to another
chain, creating an a-1,6-bond. Both starch synthases and
branching enzymes exist as multiple isoforms, thought to have
different specificities [3–5]. A third class, the debranching
enzymes, are involved in the removal of a-1,6-bonds. This
hydrolytic step is obviously required during starch re-mobilization,
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but is interestingly also required for normal starch synthesis [5–8].
Debranching enzymes can be divided into two classes: isoamylase
(ISA) and limit-dextrinase (LDA). The isoamylase class is
subdivided into proteins designated as ISA1, ISA2 and ISA3
[9,10]. ISA3 and LDA are mainly involved in starch breakdown
[11–13], whereas ISA1 and ISA2 participate in starch synthesis.
Current evidences suggest that ISA1 is an active enzyme, whereas
ISA2 proteins are non-catalytic due to changes in 6 out of 8 key
amino acids within the active site [9,14,15]. In all plant species
studied, ISA1 and ISA2 are found in heteromultimeric complexes,
in which the ISA2 subunit is proposed to have a regulatory
function or confer substrate specificity (Arabidopsis: [16]; Potato:
[17]; Chlamydomonas reinhardtii: [18]; maize: [19]; rice: [20]). In
addition, ISA1 homomultimers have been shown to occur in rice
and maize endoperm [19,20] and proposed for C. reinhardtii [18].
Thus far, homomultimer forms of ISA1 have not been reported in
leaves. Therefore, it is still unknown whether this reflects a tissue-
specific feature (heterotrophic versus autotrophic) or an evolution-
ary difference between monocot and dicot plants.
The role of these isoamylase complexes in starch biosynthesis
has been determined through phenotypic analysis in a range of
plant species and tissues in which ISA1 or ISA2 gene expression has
been reduced or abolished. The loss of ISA1 results in a reduction
of granular starch in endosperms of maize [21], rice [6] and barley
[22], in C. reinhardtii cells [18,23,24], in Arabidopsis leaves [13,16]
and in potato tubers [17]. In all instances, the starch was partially
replaced by a water soluble glucose polymer called phytoglycogen,
which had shorter chain lengths compared to amylopectin in chain
length distribution (CLD) analyses, and a higher degree of
branching. The impact of mutations in ISA2 is more variable. In
endosperms of maize and rice, loss of ISA2 had no measurable
effect, explained by the fact that these tissues contain still the active
ISA1 homomultimeric complexes [19,25]. In Arabidopsis, loss of
ISA2 causes the same phenotype as the loss of ISA1 [13,16]. This
observation is explained by the fact that Arabidopsis leaves
appears to contain only the heteromultimeric ISA1/ISA2 com-
plex, and the loss of either protein subunit results in a loss of the
enzymatic activity destabilization of the remaining subunit [15].
Overall, the differing phenotypic severity caused by the loss of
ISA1 and the existence of both homomultimeric ISA1 and
heteromultimeric ISA1/ISA2 complexes means that function of
isoamylases in amylopectin biosynthesis is still not fully under-
stood.
In this study, we wanted to address three hypotheses arising
from our current knowledge. First, are differences in starch
structure between different plant species due to different catalytic
specificities of ISA1 and ISA2 protein complexes? Second, are the
functions between the ISA complexes from different plant species
conserved? Third, are the subunits of the ISA complexes
adequately conserved such that they are interchangeable between
species? To answer these questions we replaced the endogenous
ISA proteins in the dicot Arabidopsis (AtISA1 and AtISA2) with
the respective monocot rice isoforms (OsISA1 and OsISA2). Our
data show that active heteromultimeric complexes can be formed
between AtISA1 and OsISA2 as well as AtISA2 and OsISA1.
Additionally, we were able to reconstitute the OsISA1 homo-
multimeric complex as found in rice endosperm. The resulting
transgenic lines synthesize comparable amounts of starch to wild-
type Arabidopsis plants. This illustrates that rice isoforms can
replace the endogenous Arabidopsis proteins. Furthermore, in
addition to playing a major role in facilitating starch biosynthesis,
differences in the final structure of amylopectin between the
transgenic lines suggests that the isoamylases enzymes composed of
ISA1 and ISA2 subunits also contribute to the species-specific
differences in amylopectin structure.
Results
Transient and storage starch structure in monocot anddicot plants
Despite the conservation in starch biosynthetic enzymes
between species (SSs, BEs and DBEs), the reported amylopectin
structures differ considerably between different plant species and
tissues [26]. This may be due to differences in enzyme specificities
and/or relative expression levels. To identify species with
significant differences in amylopectin structure and exclude
variability resulting from the different techniques used between
different laboratories, we analysed the structure of amylopectin
from starch extracted from two monocot species (maize and rice)
and two dicot species (Arabidopsis and potato). Although the
plants were grown under different conditions appropriate for each
species (see Material and Methods), the starch analysis was done in
parallel. We used the well-established method of chain length
distribution (CLD) analysis, where the a-1,6-bonds of amylopectin
are enzymatically hydrolysed, resulting in linear chains that can be
separated and quantified by HPAEC-PAD (high performance
anion exchange chromatography with pulsed amperometric
detection). The CLD profiles had distinct species-specific features
and varied between tissues (Fig. 1).
In the CLDs of leaf starch from all four species, chains with a
degree of polymerisation (d.p.) of 12 were most abundant, but the
profiles differed in other ways. The CLD of potato, had a
prominent peak of chains of d.p. 6 and d.p. 7 (Fig. 1C), which was
also visible in Arabidopsis, but less prominent in maize and rice
(Fig. 1). The Arabidopsis leaf starch CLD also had a discontinuity
at d.p. 18, suggestive of a subpopulation of chains of between d.p.
5 and d.p. ,20, overlapping with a population of longer chains up
to d.p. 35. This CLD discontinuity was also visible for potato leaf
starch but much less apparent for the monocot leaf starches.
Similarly, the amylopectin structure of storage starch from the
endosperms of rice and maize, and from potato tuber differed
from one another (Fig. 1B-D). Potato amylopectin still had an
early peak of chains at d.p. 6 and d.p. 7. This was also visible in
maize, but not in rice. Otherwise, the amylopectin CLDs from the
storage starches had smooth distributions up to d.p. ,35, with no
discontinuity at d.p. 18. None of the three crop species had the
same amylopectin structure within their transient and storage
starches (Fig. 1B-D). These structural differences between species
and tissues indicate that the biosynthetic machinery in each case is
not identical.
Generation of transgenic Arabidopsis lines expressingOsISA1 and OsISA2
We investigated the role of the isoamylases (ISA1 and ISA2) in
determining the starch structure between plants using a gene-swap
experiment. We chose the rice, which had the largest difference in
starch structure compared to Arabidopsis (Fig. 1). The latter is
highly amenable to genetic transformation and mutants in ISA1
and ISA2 have been characterised, together with the double
mutant [13,16].
We cloned the protein-coding region of OsISA1 and OsISA2 into
the pB7WG2 plant Gateway-compatible expression vector [27].
OsISA2 was expressed in the Arabidopsis isa2 single mutant under
the cauliflower mosaic virus (CaMV) 35S promoter. The OsISA1
construct was transformed into the Arabidopsis isa1 single mutant
and the isa1isa2 double mutants. In contrast to OsISA2, which can
only form a heteromultimeric complex with OsISA1, the OsISA1
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protein is reported to also form a homomultimeric complex [20].
Therefore, we hypothesized that the expression of OsISA1 might
be able to complement both of the missing Arabidopsis proteins.
The three Arabidopsis mutants used for transformation all have
the same reduced starch, phytoglycogen-accumulating phenotype,
due to the complete loss of the heteromultimeric isoamylase
activity. The altered glucan composition of these mutants makes it
possible to distinguish between them and wild-type plants by
simple iodine staining. Amylopectin of wild-type plants stain dark
brown whereas the phytoglycogen in the mutants stains a more
red-brownish colour (Fig. 2A). We isolated a minimum of ten
independent lines per transformation from the T1 generation. We
then screened these lines by iodine staining to search for those in
which the loss of the endogenous isoamylase activity was
complemented by the expression of the rice gene. Half or more
of the T1 transformants stained like the wild type, suggesting
complementation. We confirmed that this phenotype was stable in
the T2 generation of the three most promising candidate lines per
genotype (Fig. 2A). These lines were analysed further and were
named as follows: isa1 mutants transformed with OsISA1, isa1-
35S::OsISA1 A, B and C; isa2 mutants transformed with OsISA2,
isa2-35S::OsISA2 A, B and C; isa1isa2 double mutants transformed
with OsISA1, isa1isa2-35S:: OsISA1 A, B and C.
Quantitative starch and phytoglycogen measurementIodine staining is suitable for rapid screening but is largely
qualitative. Therefore we measured the starch and phytoglycogen
in all of the transformed lines at the end of a 12-h photoperiod. As
expected, wild-type plants had around 8 mg starch per gram plant
fresh weight and contained only trace amounts of soluble glucan.
In contrast, the isa1, isa2 and isa1isa2 mutants contained both
starch and phytoglycogen and synthesized less glucan in total than
the wild type (Fig. 2B). All three mutants had the same phenotype,
as reported earlier [13,16]. The starch measurements for the three
isa1-35S::OsISA1 lines confirmed the results from the iodine
staining (Fig. 2A) as they contained wild-type levels of starch
and no phytoglycogen. Interestingly, full complementation was
also achieved in the isa1isa2-35S::OsISA1 plants despite the fact
that no ISA2 protein is present (neither AtISA2 nor OsISA2).
Comparable results were also recently described for these isa
mutants transformed with the Maize ISA1 gene [28].
The three isa2-35S::OsISA2 lines showed a marked change in
phenotype compared with the parental line, but complementation
was incomplete. Although all three lines contained only starch,
with little or no phytoglycogen, the starch content varied
significantly between the lines (Fig. 2B). The line isa2-35S::OsISA2
B contained only half as much starch as the wild-type (comparable
to the sum of starch and phytoglycogen in the isa2 parental line),
Figure 1. Amylopectin structure in different plant species and tissues. Comparison of chain length distributions (CLDs) of starch in differenttissues from Arabidopsis thaliana, Oryza sativa (rice), Solanum tuberosum (potato) and Zea mays (maize). A) CLD of Arabidopsis leaf starch, harvestedat the end of the light period. B) CLDs of rice leaf starch (filled grey circle) and seed storage starch (open grey circle). C) CLDs of potato leaf starch(filled red circle) and tuber storage starch (open red circle). D) CLDs of maize leaf starch (filled blue circle) and seed storage starch (open blue circle).Note that in all cases, the CLDs differ between the tissues from the same plant species. Values are means 6 SE (n = 3).doi:10.1371/journal.pone.0092174.g001
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whereas isa2-35S::OsISA2 C contained almost wild-type amounts
of starch.
Rice isoamylase complex formation in ArabidopsisThe complementation of the Arabidopsis mutants suggests that
the rice proteins are functional and can substitute the loss of the
endogenous proteins. To validate this further we used native
PAGE with gels containing beta-limit dextrin, a good substrate to
visualize isoamylase enzyme activity. The three isoamylase
mutants, isa1, isa2 and isa1isa2 lacked the debranching activity
found in wild-type plants (Fig. 3 and [16]). In isa1isa2-35S::OsISA1
lines two new activities were visible compared with the isa1isa2
parental line (Fig. 3B). This is consistent with the idea that two
homomultimeric forms of OsISA1 with different electrophoretic
mobilities are formed [29]. Interestingly, in isa1-35S::OsISA1 lines,
three new enzyme activities were visible compared with the isa1
parental line. Two migrated at positions comparable to the
activities observed when OsISA1 was expressed in the isa1isa2
double mutant (i.e. likely homomultimers of OsISA1) while the
third ran at the same height as the endogenous Arabidopsis ISA1/
ISA2 complex (Fig. 3A). This is most likely a chimeric isoamylase
heteromultimer comprised of OsISA1 and AtISA2 subunits.
Surprisingly, in the isa2-35S::OsISA2 lines, no additional enzyme
activities were detected compared to the isa2 parental line. This
was unexpected because even though there was phenotypic
variability, these plants clearly differed from the isa2 mutants in
respect to starch and phytoglycogen content and more closely
resembled the wild type (See Fig. 2).
Effect of the rice isoamylases on endogenous isoamylaseprotein levels
In Arabidopsis, only the heteromultimeric ISA1/ISA2 complex
occurs and, in mutant plants lacking either ISA1 or ISA2, the
activity is abolished (see Fig. 3). Furthermore, evidence suggests
that the remaining protein is unstable in the absence of its partner.
Thus, ISA1 protein is strongly reduced in amount in isa2 mutants
and ISA2 protein is undetectable in isa1 mutants [15,16]. Given
this profound effect of one protein on the stability of its interaction
partner, we tested what impact the expression of the rice
isoamylases has on the levels of the endogenous Arabidopsis
proteins.
The same protein extracts used for the native PAGE were
analysed by SDS-PAGE and immunoblotting, probing with
specific antibodies raised against AtISA1 [16] and AtISA2 [15].
We confirmed that the ISA1 protein was reduced in isa2 mutants
and the absence of ISA2 protein in isa1 (Fig. 3 D and E). In isa1-
Figure 2. Starch content in Arabidopsis isoamylase mutants transformed with rice isoamylases. A) Arabidopsis T1 transformed linesexpressing rice ISA1 (OsISA1) or rice ISA2 (OsISA2) in the background of isa1, and isa2 single and isa1isa2 double mutants. Qualitative iodine stainingwas used to isolate plants which revert the red-brown staining of the isa1, isa2, isa1isa2 mutants back to wild-type (WT) staining. Three independentlines are shown (A, B and C), which were used in all subsequent experiments. B) Plants grown for 28 days in a 12-h light/12-h dark regime wereharvested at the end of the day and analysed for insoluble starch (grey bar) and phytoglycogen (white bar). Arabidopsis isa1 mutants complementedwith OsISA1 (isa1-35S::OsISA1), isa2 mutants complemented with OsISA2 (isa2-35S::OsISA2) and isa1isa2 double mutants complemented with OsISA1alone (isa1isa2-35S::OsISA1). Samples comprised all the leaves from a single plant and were extracted using perchloric acid. Starch in the insolublefraction and phytoglycogen in the soluble fraction were measured after enzymatic hydrolysis to glucose. Each point is the mean 6 SE (n = 4).doi:10.1371/journal.pone.0092174.g002
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35S::OsISA1 lines, the Arabidopsis ISA2 protein reappeared,
although not to wild-type levels (Fig. 3D). This observation is in
agreement with the native PAGE (Fig. 3A), where a putative
chimeric OsISA1/AtISA2 heteromultimer was detected. It is worth
noting that the activity of this putative chimeric enzyme was less
intense than that of the endogenous ISA1/ISA2, consistent with
the lower levels of AtISA2 protein. Nevertheless, these observations
strongly suggest that, in these plants, the OsISA1 protein interacts
with and stabilises the Arabidopsis ISA2 protein.
In contrast, no increase in the AtISA1 protein could be detected
in isa2-35S::OsISA2 compared to the isa2 parental line (Fig. 3E). It
is possible that the residual AtISA1 in isa2 is available to form an
active chimeric heteromultimer composed of AtISA1 and OsISA2
subunits. This would explain the observed partial complementa-
tion of the isa2 phenotype in these lines (Fig. 2B). However, our
inability to detect isoamylase activity in these lines renders this
explanation somewhat speculative.
Starch structure in Arabidopsis isoamylase mutantsexpressing rice isoamylases
In addition to the accumulation of phytoglycogen, loss of the
ISA1/ISA2 isoamylase in Arabidopsis results in alterations of the
CLD of the residual starch (Fig. 4A and [13,16]). We analysed
starch from plants harvested at the end of the day (the same
samples as used for the starch measurements; Figure 2B). CLD
profiles were calculated by averaging the three biological replicates
of each genotype. As previously reported, the amylopectin CLDs
from the starch from all three Arabidopsis isa mutants (isa1, isa2
and isa1isa2) were the same, having more very short chains (d.p. 4
to 7) but less medium-length chains (d.p. 10 to 16) compared to the
wild type (only isa1isa2 is shown). We compared the amylopectin
CLDs of the starch synthesized in our Arabidopsis mutants
complemented with the rice isoamylases to the wild type, to the
isa1isa2 double mutant, and to the rice starches. For each
transformation, the three independent lines had very similar
CLD profiles (Fig. S1). Both the isa1-35S::OsISA1 and the isa2-
35S::OsISA2 lines synthesized starch more similar to wild-type
Arabidopsis starch than to isa1isa2 mutant (Fig. 4 B and D).
Interestingly, the isa1isa2-35S::OsISA1 CLD profile was very
different from the isa1isa2 mutant, and was intermediate between
wild type Arabidopsis starch and rice leaf starch (Fig. 4C).
Discussion
Interchangeability of rice and Arabidopsis isoamylasesubunits
In this work, we demonstrated the interchangeability of
enzymes in starch metabolism between monocotyledonous and
dicotyledonous plants. Arabidopsis mutants lacking the ISA1/
ISA2 complex have reduced capacity to synthesise crystalline
starch - instead they accumulate water-soluble phytoglycogen.
Here, we show that the rice isoamylase proteins are able to rescue
this phenotype. All the transgenic lines analysed contained
exclusively starch (Fig. 2B), even though the responsible isoamylase
complex differed in each case. In isa1isa2 expressing OsISA1, we
observed two complexes (Fig. 3B). In these plants OsISA1 is the
sole ISA protein present in these plants, suggesting that these two
are homomultimeric OsISA1 complexes. The formation of an
OsISA1 complex was expected, as a homo-pentameric form in rice
Figure 3. Reconstitution of isoamylase complexes in Arabidopsis isoamylase mutants expressing OsISA1 and OsISA2. A) Solubleprotein extracts of the wild type (WT), isa1, isa2, and isa1isa2 mutants, and two isa1 lines complemented with OsISA1 (isa1-35S::OsISA1 A and B),analyzed by native PAGE in beta-limit dextrin-containing gels. After electrophoresis and incubation, gels were stained with iodine to reveal bands ofactivity where the glucan was hydrolyzed. White arrows; the endogenous heteromultimeric Arabidopsis isoamylase (AtISA1/AtISA2). Black and whitearrow; the chimeric Arabidopsis/rice isoamylase (OsISA1/AtISA2). Black arrows; the homomutlimeric rice isoamylase (OsISA1). B) Soluble proteinextracts of controls (as in A) and two isa1isa2 lines complemented with OsISA1 (isa1isa2-35S::OsISA1 A and B). Arrows as described in A. C) Solubleprotein extracts of controls (as in A) and two isa2 lines complemented with OsISA2 (isa2-35S::OsISA2 A and B). Arrow as described in A. D) AtISA2(arrow), detected by immunoblotting (same sample order as in A). Note that AtISA2 is not detectable in isa1, isa2, isa1isa2 mutants, but is visible inthe transformed lines. E) AtISA1 (arrow), detected by immunoblotting (same sample order as in C). Note that AtISA1 amount is reduced in the isa2mutant, but does not increase in the transformed lines.doi:10.1371/journal.pone.0092174.g003
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endosperm has been reported [20]. The observation of two
complexes in our plants suggests that the OsISA1 protein can also
adopt a different oligomeric state, or is post-translationally
modified in a way that affects its migration in our gels (i.e. by
changing surface charge or substrate affinity). Consistent with our
observations, early studies in which the homomultimer was
purified to homogeneity from rice endosperm also yielded an
enzyme which migrated in several positions on a native gel and in
two positions during isoelectric focusing [29]. Importantly, these
data show that starch synthesis in Arabidopsis is not dependent on
the presence of ISA2, even though this subunit is critical for the
stability of the endogenous enzyme.
In the isa1 lines expressing OsISA1, we observed an additional
enzymatic activity besides the two homomultimeric OsISA1
complexes (Fig. 3A). The difference compared to isa1isa2
expressing OsISA1 is the intact AtISA2 gene, so the additional
activity is most probably a chimeric heteromultimeric isoamylase
complex composed of OsISA1 and AtISA2 subunits. This is in
agreement with the increased AtISA2 protein content in these
lines, compared to the isa1 mutants, in which AtISA2 protein is not
detectable (Fig. 3D; [15]). The formation of a complex between
OsISA1 and AtISA2 presumably stabilizes the AtISA2 protein. This
result implies that the ISA1 and ISA2 proteins are sufficiently
conserved between the dicot Arabidopsis and the monocot rice to
allow assembly into an enzymatically functional complex. This is
consistent with the ability of the maize ISA1 to form an active
chimeric complex with AtISA2 in vitro [28].
In the isa2 lines expressing OsISA2, the isa2 phenotype was
rescued and the plants produced exclusively starch and no
phytoglycogen. The most likely explanation for this would be that
AtISA1 and OsISA2 assemble into an active enzyme in these
plants, as seen for OsISA1 and AtISA2. However, unexpectedly,
we were not able to detect an additional isoamylase activity
compared to isa2 mutants (Fig. 3C). It is possible that the activity
may be below the detection limit of our native gel assay. Although
we tried increasing the protein content five-fold and prolonging
the incubation time from 2 h to 12 h, no activity band was
observed even under these conditions. We also did not observe a
Figure 4. Complementation of the altered amylopectin structure in Arabidopsis isoamylase mutants transformed with riceisoamylases. Starch was extracted from replicate plant samples (as used in Fig. 2B), and the amylopectin CLDs were determined (see Materials andMethods and Fig. 1). The difference plots presented were calculated by subtracting the relative percentage for each chain length of Arabidopsis wild-type amylopectin from the equivalent percentage for the amylopectin from the lines indicated. Errors are calculated from the SE of two comparedsamples, considering the error law of Gauss. A) Difference plots for amylopectins from isa1isa2 (blue diamond), rice transient starch (filled black circle)and rice storage starch (open black circle). Note that the CLD of isa1isa2 amylopectin resembles those of isa1 and isa2 single mutants (not shown).Therefore the isa1isa2 difference plot and that of rice transient starch are used as references in panels B to D. B) Difference plots of isa1-35S::OsISA1lines relative to the wild type (red triangle). C) Difference plots of isa1isa2-35S::OsISA1 lines relative to the wild type (red triangle). D) Difference plotsof isa2-35S::OsISA2 lines relative to the wild type (red triangle). Note that in B to D, the red lines do not simply revert to zero, which would beequivalent to wild-type Arabidopsis amylopectin. They differ more from isa1isa2 amylopectin (blue diamond) and, to an extent, approach the CLD ofrice transient starch (filled black circle).doi:10.1371/journal.pone.0092174.g004
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stabilising effect of OsISA2 expression on AtISA1 protein levels (in
contrast to the stabilisation of AtISA2 by overexpressing OsISA1).
However, there is residual AtISA1 protein in the isa2 mutants,
which was comparable in lines overexpressing OsISA2. This might
mean that there is sufficient protein to partner OsISA2. It is also
possible that a chimeric complex is formed which has an altered
migration such that it co-migrates with another starch degrading
activity and is therefore masked. Alternatively, the chimeric
enzyme may not resolve well under our experimental conditions or
may be very labile during extraction.
The idea that a chimeric AtISA1/OsISA2 isoamylase complex
may be present at a very low abundance in our isa2-35S::OsISA2
plants would suggest that extremely low levels of a functional
isoamylase complex are sufficient to facilitate starch synthesis.
Although all three lines had a similar starch structure it is worth
noting that the starch level was not the same as in the wild type
(Fig. 2B). The fact that very low levels of isoamylase may be
functional was also suggested from a study of potato plants in
which either StISA1 or StISA2 were silenced. Transgenic lines
were identified which showed no detectable isoamylase activity
on native PAGE (as used here) but still accumulated almost
exclusively starch and only tiny amounts of phytoglycogen [17].
This study differs from most, as intermediate levels of the wild-
type isoamylase activity were obtained. In contrast, when mutants
are used, they generally result in a complete loss of isoamylase
activity or an altered isoamylase enzyme, which is typically
accompanied by a phytoglycogen-accumulating phenotype (e.g.
[6,16,18,21,22]. These results lead us to speculate that as
isoamylase activity is reduced and becomes limiting, the initial
phenotype may be a drop in starch production before further
reduction in the enzyme results in phytoglycogen accumulation.
However, this is an aspect of the phenotype that we cannot fully
explain at present.
The fact that the rice proteins were able to functionally replace
the Arabidopsis proteins does not necessarily mean that the
converse is true, and that the Arabidopsis proteins would be able
to complement the phenotypes of the corresponding rice mutants.
This may be especially the case for ISA1, because Arabidopsis
ISA1 seems unable to form an active homomultimer. This may
be important as it is specifically the ISA1 homomultimer that is
thought to facilitate starch synthesis in the rice endosperm [25].
Mutants lacking ISA2 have only the homomultimer and produce
near-normal starch. In contrast, plants overexpressing ISA2
possess the heteromultimer, but lack the ISA1 homomultimer.
These plants produce only half as much starch as the wild type
and the starch is aberrant in structure, suggesting that the
specificity of the heteromultimer is not ideally matched to the
glucan produced by the other starch biosynthetic enzymes of the
endosperm [25].
A role for isoamylase in determining species-specificstarch structure?
The replacement of Arabidopsis ISA1 and ISA2 with the rice
orthologs enabled us to study the effect of distinct isoamylase
complexes on the fine structure of starch. Given that the
isoamylases of different subunit compositions have been shown
to have distinct functionalities in cereal endosperm, it is reasonable
to hypothesise that the differences in amylopectin between species
may be partly dependent on the isoamylases they contain. All
three transgenic lines had starch structures that were similar, but
not identical, to Arabidopsis wild-type starch (Fig. 4). The line
possessing only the rice homomultimers had the most divergent
amylopectin structure. The increased abundance of short chains
between d.p. 3 and d.p. 8 in the isa1isa2 mutant CLD (compared
to wild-type Arabidopsis amylopectin) was lost. Instead an
increased abundance of longer chains between d.p. 6 and d.p.
14 was seen. This structure is somewhat intermediate between
Arabidopsis and rice transient starch. In the isa1-35S::OsISA1 lines
the amylopectin CLD was much more similar to wild-type
Arabidopsis amylopectin, but the same tendencies towards
increased number of chains between d.p. 6 and d.p. 14 was seen.
In this line, the homomultimeric OsISA1 is accompanied by a
chimeric Arabidopsis/rice isoamylase complex - OsISA1/AtISA2.
This chimeric enzyme presumably functions in a similar way to the
endogenous Arabidopsis enzyme. Plants putatively containing the
other chimeric Arabidopsis/rice isoamylase complex - AtISA1/
OsISA2 also had starch with modest increases in the numbers of
chains between d.p. 6 and d.p. 14. It has been proposed that the
catalytically inactive subunit ISA2 has a regulatory function,
provides substrate specificity, or increased stability to the catalytic
ISA1 subunit [5]. These results suggest that the replacement of the
AtISA2 with OsISA2 causes a change in substrate specificity, but
this conclusion needs to be treated with caution since no activity
was directly measured. Further analysis, such as X-ray scattering,
scanning electron microscopy and differential scanning calorim-
etry could be used to study in more depth the structure and
properties of starch produced in plants with different chimeric
isoamylase complexes.
Conclusion
Our results are consistent with the findings of other studies.
The wheat ISA1 (TaISA1) protein was able to revert the
phytoglycogen-accumulating phenotype of the rice sugary-1
mutant (lacking OsISA1) back to a starch-accumulating pheno-
type [30]. However, rice and wheat are much more closely
related to each other than rice and Arabidopsis used, and wheat
may also have the two types of isoamylase complex (i.e. homo-
and heteromultimers). Nevertheless, after complementation with
TaISA1, the amylopectin CLD was similar – but not identical –
to the wild type rice amylopectin. Recently, it was shown that the
maize ISA1 (ZmISA1) protein expressed in the Arabidopsis isa1
and isa1isa2 mutants were able to restore exclusive starch
synthesis in Arabidopsis, similar to our observations. Interesting-
ly, the authors observed a greater variability of the starch content,
with some transgenic lines producing even more starch than the
wild type, which was not the case in our plants. However, this
raises the possibility that the isoamylase may somehow deter-
mines not only the starch structure but also the amount
produced. Interestingly, the ZmISA1 expressing plants produced
starch with the same amylopectin CLD as the Arabidopsis wild
type [28]. This is in contrast to our results, but might be
explained by the fact that the amylopectin CLDs of maize and
Arabidopsis are more similar to each other than Arabidopsis and
rice (Fig. 1). It is tempting to conclude from all of these studies
that isoamylase can contribute to species-specific amylopectin
structure. However, it is important to note that the differences in
structure that we measure are relatively small. Therefore, it seems
likely that species-specific differences in the starch synthases and
branching enzymes might also contribute.
Furthermore, it is important to emphasise that differences in
expression of these enzymes between tissues or between species
may have an equally large impact on the final structure of
amylopectin. This was strikingly illustrated recently through
RNA-seq analysis of the myrmecophytic tropical tree Cecropia
peltata, where differential expression of the starch-biosynthetic
apparatus results in amylopectin production in leaves and
Rice Isoamylases Replacing Arabidopsis Enzymes
PLOS ONE | www.plosone.org 7 March 2014 | Volume 9 | Issue 3 | e92174
glycogen biosynthesis in food bodies produced to feed mutualistic
ants [31].
Materials and Methods
Plants and growth conditionsArabidopsis thaliana plants were grown in Percival AR95 climate
chambers at constant 20uC, 60% relative humidity, 12-h
photoperiod and a light intensity of 150 mmol photons m22
sec21. Seeds were sown out on soil and seedlings transferred to
individual pots two weeks after germination. Mature plants were
harvested three weeks later, weighed, frozen in liquid N2 and kept
at –80uC until use. Single isa mutants used were described in
earlier studies: isa1, isa1-1, SALK_042704 [16]; isa2, isa2-1
(initially called dbe1-1 [32] and the double mutant isa1isa2, isa1-
1/isa2-1 [16].
Rice (Oryza sativa cv. Taipei 309) plants were grown in
greenhouse at 23uC, 70% relative humidity at night and 26uC,
80% relative humidity at day with a minimum 14-h photoperiod
and minimum light intensity of 250 mmol photons m22 sec21.
Leaves for the extraction of transient starch were harvested from
four-week-old plants. Mature, dry seeds from these plants were
used for storage starch purification. Potato (Solanum tuberosum cv.
Agata) tubers were obtained from a local market and used for
storage starch purification. For transient starch isolation, tubers
were sprouted and leaves harvested after four weeks of growth in a
chamber (Kalte 3000 AG, Landquart, Switzerland) at constant
22uC, 70% relative humidity, with a 12-h photoperiod and a light
intensity of around 250 mmol photons m22 sec21. Maize (Zea mays
Benicia, Pioneer) plants were grown in a chamber (Kalte 3000
AG) at constant 22uC, 70% relative humidity with a 12-h
photoperiod and a light intensity of around 250 mmol photons
m22 sec21. Leaves for transient starch were harvested from four-
weeks-old plants. Purchased seeds were used directly for storage
starch purification.
SDS-PAGE blotting and native PAGESoluble proteins were extracted from leaves (150 mg) of five-
week-old plants by homogenization in 1 mL of medium contain-
ing 100 mM MOPS, pH 7.2, 1 mM EDTA, 5 mM DTT, 10%
(v/v) glycerol, 1 x Complete Protease Inhibitor Cocktail (Roche),
using all-glass homogenizers. Homogenates were subject to
centrifugation (16,000 g, 10 min, 4uC). Proteins were separated
by SDS-PAGE using standard methods, transferred to PVDF
membranes, probed with antibodies against AtISA1 [16] and
AtISA2 [15], and developed using Immun-Star horseradish
peroxidase chemiluminescence (Bio-Rad). For native PAGE,
proteins were separated with gels containing 6% (w/v) polyacryl-
amide and 0.3% (w/v) b-limit dextrin (Megazyme, Bray, Ireland).
After electrophoresis, gels were incubated for 2 h at 37uC in
medium containing 100 mM Tris-HCl, pH 7.2, 1 mM MgCl2,
1 mM CaCl2 and 5 mM DTT. Reaction were stopped and
activities visualized by application of Lugol solution (5% [w/v] I2,
10% [w/v] KI).
Extraction and measurement of glucose polymersThe extraction procedure was the same for all plant species and
tissues and was performed using perchloric acid as described
previously [16]. Glucose polymers (starch and phytoglycogen)
were quantified by enzymatic digestion with amyloglucosidase
(Aspergillus niger; Roche) and a-amylase (pig pancreas; Roche), and
released glucose was measured spectrophotometrically [33].
Structural analysis of starch and phytoglycogenStarch and phytoglycogen in the insoluble and soluble fractions
from the perchloric acid-extracted plant material, respectively,
were used for glucan structural analysis. Volumes containing
100 mg of starch or phytoglycogen were used from each of four
individually extracted plants. Samples were boiled for 10 min prior
enzymatic digestion. Debranching reactions with Pseudomonas
amyloderamosa isoamylase (Sigma-Aldrich, Buchs, Switzerland) and
Klebsiella planticola pullulanase (Megazyme) were carried out for 2 h
at 37uC in 10 mM Na-acetate, pH 4.8. The resulting linear
glucans were analysed by HPAEC-PAD as described previously
[8].
Construction of OsISA1 and OsISA2 overexpressionvectors and plant transformation
OsISA1 and OsISA2 coding sequences were amplified by PCR
using attB-site containing primers (Table S1). The templates were
pGEM-T Easy vectors containing OsISA1 and OsISA2 cDNA
sequences [25]. The PCR fragments were cloned into pDONR
221 via the BP reaction of the Gateway recombination cloning
technology (Invitrogen, LuBioScience GmbH. Lucerne, Switzer-
land). After sequence conformation, inserts were cloned into the
plant vector pB7WG2 [27] using the Gateway LR reaction. Stable
Arabidopsis thaliana transgenic lines were generated through
Agrobacterium tumefaciens-mediated transformation using the floral
dip method [34]. Independent transformants were selected on soil
by spraying two-week-old plants with 0.1% (v/v) BASTA
herbicide.
Supporting Information
Figure S1 Amylopectin structure of starch produced inindependent isa1-35S::OsISA1, isa2-35S::OsISA2 andisa1isa2-35S::OsISA1 lines. Comparison of the three inde-
pendent transformants lines of isa1-35S::OsISA1, isa1isa2-
35S::OsISA1 and isa2-35S::OsISA2 illustrating the uniformity in
starch structure produced. Raw data used to calculate the mean
normalized CLD (6 SE, n = 3) was also used to calculate the
difference plots in Fig. 5 B to D. A) CLDs of the lines isa1-
35S::OsISA1 A (open circle), isa1-35S::OsISA1 B (open square) and
isa1-35S::OsISA1 C (open triangle). B) CLDs of the lines isa1isa2-
35S::OsISA1 A (open circle), isa1isa2-35S::OsISA1 B (open square)
and isa1isa2-35S::OsISA1 C (open triangle). C) CLDs of the lines
isa2-35S::OsISA2 A (open circle), isa2-35S::OsISA2 B (open square)
and isa2-35S::OsISA2 C (open triangle).
(TIF)
Table S1 Primers used for Gateway cloning of OsISA1and OsISA2.
(DOCX)
Acknowledgments
We thank our trainee technicians Markus Reichlin and Raphaela
Gschwind and our under-graduate student Stefan Oberlin for experimental
help. Furthermore, we thank Yasunori Nakamura for the full length clones
of OsISA1 and OsISA2.
Author Contributions
Conceived and designed the experiments: SS. Performed the experiments:
SS. Analyzed the data: SS. Contributed reagents/materials/analysis tools:
SS SCZ. Wrote the paper: SS SCZ.
Rice Isoamylases Replacing Arabidopsis Enzymes
PLOS ONE | www.plosone.org 8 March 2014 | Volume 9 | Issue 3 | e92174
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